npg CBP/p300 and lung 324 Cell Research (2007) 17: 324-332 npg © 2007 IBCB, SIBS, CAS All rights reserved 1001-0602/07 $ 30.00 REVIEW www.nature.com/cr

Roles of CREB-binding protein (CBP)/p300 in respiratory epithelium tumorigenesis

Michalis V Karamouzis1, Panagiotis A Konstantinopoulos1, 2, Athanasios G Papavassiliou1

1Department of Biological Chemistry, Medical School, University of Athens, 75, M Asias Street, 11527 Athens, Greece; 2Division of Hematology-Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA

CREB-binding protein (CBP) and its homologue p300 are transcriptional co-activators of various sequence-specific factors that are involved in a wide array of cellular activities, such as DNA repair, , differentia- tion and apoptosis. Several studies have suggested that CBP and p300 might be considered as tumour suppressors, with their prominent role being the cross-coupling of distinct expression patterns in response to various stimuli. They exert their actions mainly via acetylation of and other regulatory proteins (e.g. ). A major paradox in CBP/ p300 function is that they seem capable of contributing to various opposed cellular processes. Respiratory epithelium tumorigenesis represents a complex process of multi-step accumulations of a gamut of genetic and epigenetic aberra- tions. Transcription modulation through the alternate formation of activating and repressive complexes is the ultimate converging point of these derangements, and CBP/p300 represents key participants in this interplay. Thus, illumination of their molecular actions and interactions could reveal new potential targets for pharmacological interventions in re- spiratory epithelium carcinogenesis. Keywords: CBP, p300, lung cancer, acetylation, Cell Research (2007) 17:324-332. doi: 10.1038/cr.2007.10; published online 20 March 2007

Introduction fects, albeit abnormal heart formation was noted only in p300–/– mice, thus suggesting that both proteins are cAMP response element-binding protein-binding pro- necessary during embryogenesis with overlapping and tein (CBP) and p300 were originally identified as factors unique functions [6]. Consistently, double heterozygous binding to the cAMP response element-binding protein CBP+/– and p300+/– knockout mice are also embryonic (CREB) and the adenoviral E1A, respectively [1, 2]. The lethal. However, only CBP+/– mice display features of human CBP resides in the chromosomal region Rubinstein-Taybi syndrome (RTS) [6, 7], while a more 16p13.3 and shows homology to 22q13, where p300 is severe and penetrant RTS-like phenotype was found in located [3]. CBP/p300 proteins share several conserved mice in which one CBP allele was modified to express a regions, which constitute most of their known functional truncated CBP protein [7]. domains (Figure 1). CBP and p300 have interchangeable Apart from other structurally defined regions, CBP/p300 roles during embryonic development and in many processes have specific areas for interaction with a wide array of tran- govern cellular homeostasis [4, 5]. However, genetic and scription factors and co-factors (Figure 1). The plethora of molecular evidence suggests that they also fulfil distinct these interacting proteins indicates the unique involvement functions [3]. Homozygous CBP–/– and p300–/– knock- of CBP/p300 in transcriptional control as ubiquitous and out mice were inviable due to severe developmental de- versatile co-integrators. Many of the protein interactions with CBP/p300 are regulated by upstream signals. For ex- ample, phosphorylation of the transcription factor CREB

Correspondence: Athanasios G Papavassiliou modulates its interaction with CBP, while hormones can Tel: +30-210-7462509; Fax: +30-210-7791207 induce the binding of CBP/p300 to nuclear receptors [8]. E-mail: [email protected] Notably, in some cases CBP/p300 can stimulate diverse

Cell Research | www.cell-research.com Michalis V Karamouzis et al. npg 325

Transactivation Acetyltransferase Transactivation COOH NH2 66% 93% 86% 86% 66% 82%

CBP 2441 a.a. BD QP KIX SID RID CH1 CH2 CH3

p300 2414 a.a. BD QP SID KIX RID CH1 CH2 CH3

Nuclear RXR CREB Elk-1 c-Fos SRC-1 receptors STAT2 c-Jun MyoD MyoD p53 HIF-1 Elk-1 E2F ACTR Ets-1 c-Myb GATA-1 Smad BRCA1 E1A p53 SREBP p53 Tax p73 p53 pCAF STAT1 SV40 large T pp90 RSK E1A p73 TBP TBP c-Jun

Figure 1 Schematic representation of CBP and p300 homologous regions and functional domains along with a selected list of proteins that bind to specific sites of CBP/p300. BD, ; CH1-3, cysteine and -rich regions 1-3; KIX, binding site of CREB; QP, glutamine- and proline-rich domain; RID, receptor-interacting domain; SID, steroid receptor co-activator-1 interaction domain.

functions of certain transcriptional regulatory proteins [4, CBP/p300 transcriptional activity 5]. Nevertheless, the most intriguing feature of CBP/p300 is their stoichiometric function in vivo and their intrinsic The multifaceted role of CBP/p300 in transcription can enzymatic activities. be achieved by various mechanisms (Figure 2). CBP/p300 The importance of CBP/p300 is underscored by the are thought to serve as a physical “bridge” between diverse fact that genetic alterations as well as their functional dys- gene-specific transcription factors (GSTFs) and compo- regulation are strongly linked to human diseases. Germline nents of the basal transcriptional machinery (BTM; e.g. of CBP were first reported in RTS, an autosomal- TATA box-binding protein, TFIIB, TFIIE, TFIIF) thereby dominant disease characterised by mental retardation, skel- stabilising the transcription complex [4]. CBP/p300 might etal abnormalities and a high malignancy risk, albeit such also act as a scaffold for the formation of multi-component defects have not been associated with p300 so far [9, 10]. complexes containing transcription factors and co-factors. Nonetheless, mutations of the p300 gene have been detected A classical example of complex assembly involving mul- in human epithelial tumours, which is consistent with the tiple transcription factors and co-factors is the β- general notion that p300 might possess tumour-suppressor gene promoter in response to viral infections [14]. The activity [11, 12]. Although the tumour-suppressor function large size of CBP/p300 endows them with many different of CBP is still unclear, its involvement in chromosomal interaction surfaces, thus enabling them to bind concur- translocations associated with haematologic malignancies rently to various proteins. By providing a platform for the has been well-documented [13]. The critical involvement of assembly of transcription regulatory proteins, CBP/p300 CBP/p300 proteins in a variety of key molecular pathways might increase the relative concentration of these factors provides the mechanistic rationale of their implication in in the local transcriptional environment (Figure 2). Ac- respiratory epithelium tumorigenesis. cordingly, cells can cooperatively utilise its repertoire www.cell-research.com | Cell Research npg CBP/p300 and lung cancer 326

CBP/p300

GSTF BTM

DNA Other 1 co-factors Post-translational CBP/p300 modifications

2 GSTF GSTF GSTF CBP/p300 DNA Auto- acetylation GSTF Ac 3 acetylation CBP/p300 acetylation Ac Ac Ac Ac Ac Ac GSTF BTM

DNA Ac Ac Ac Ac

Figure 2 CBP/p300 participate in transcriptional control through various mechanisms. (1) “Bridging” GSTFs with the BTM. (2) Contributing to the formation of multi-protein complexes and directly and/or indirectly modulating the activation status of GSTFs through post-translational modifications. (3) Exhibiting acetyl-transferase activity on and certain GSTFs. Ac, acetyl group.

of proteins, so that the combinations of a few ubiquitous to enhance DNA-binding activity and gene transcription factors, and signal- and tissue-specific modulators, could [24]. Moreover, CBP/p300 can bind additional co-factors create a broad spectrum of regulatory complexes. that possess acetyl-transferase activity (e.g. p300/CBP- Post-translational chromatin modifications modulate the associated factor (p/CAF)) [25], and also recruit proteins activity of many by modifying both the core histones bearing other chromatin-modifying enzymatic activities and non-histone transcription factors [15]. Acetylation of (e.g. histone methyltransferases) [26]. multiple sites in the histone tails has been directly associ- The ability of so many proteins to interact with CBP/p300 ated with transcriptional upregulation, while de-acetylation suggests that competition for the rather limited intracellular correlates with transcriptional repression. Mechanistically, pool of CBP/p300 might account for the observation that histone acetylation promotes the accessibility of DNA to unrelated transcription factors inhibit each other without transcription protein complexes, by facilitating the “unwir- direct interference [27, 28]. In this vein, sequestration of ing” of the chromatin structure [16]. CBP/p300 can interact CBP/p300 by the adenoviral protein E1A [29], human with chromatin nucleosomes via nucleosome assembly papilloma virus protein E6 [30] and other viral proteins proteins, histone-binding proteins and possibly histones [3] is probably a means by which oncogenic viruses sup- themselves [17, 18]. In addition to histones, CBP/p300 also press many cellular transcription factors, and thereby may modulate a variety of other proteins by acetylation [19, 20]. contribute to cellular transformation. In most instances, acetylation of transcription factors has Given the multiple activities of CBP/p300, it is of been shown to enhance their DNA-binding activity (e.g. paramount importance to enlighten their own regulatory p53, p73, retinoblastoma (Rb), E2F, Sp3, signal transducers principles. CBP/p300 are thought to be modulated by and activators of transcription) [11, 21], although it seems phosphorylation via cyclin/cyclin-dependent kinase (Cdk)

plausible that acetylation also regulates protein-protein complexes in G1/S [31], whereas various kinases, such interactions, protein-DNA recognition [19], as well as as protein kinase A, protein kinase C, phosphatidylinosi- nuclear transport and structure [22, 23]. Acetylation of tol-3 kinase/AKT and mitogen-activated protein kinases components of the BTM (e.g. TFIIE, TFIIF) has been found (MAPK), have been shown to phosphorylate CBP in vitro

Cell Research | www.cell-research.com Michalis V Karamouzis et al. npg 327

[19, 32]. Furthermore, CBP/p300 are also targets of other CBP/p300 in lung carcinogenesis post-translational modifications, such as methylation [33] and sumoylation [34]. Recent data have indicated the CBP/p300 “preferential” existence of an auto-regulatory loop, whereby the acetyl- usage by oncogenic transferase activity of p300 is considered to be intrinsically GSTFs CBP/p300 over-expression CBP/p300 weak [35], and its auto-acetylation, or possibly acetylation Genetic aberrations sequestration via other proteins, might stimulate its acetyl-transferase causing CBP/p300 by oncogenic activity [35]. De-acetylases (e.g. 1 loss of funcion viral proteins (HDAC1)) or other proteins (e.g. p53) could associate with p300 to keep it in a catalytically inactive state [36, 37]. On Other co-factors the other hand, it has been shown that p300 can also inacti- CBP/p300 vate HDAC1 via acetylation. Thus, two positive feedback Nucleosome loops (activation through auto-acetylation and inhibition of GSTF BTM an inhibitor) ensure maximal p300 activity. A recent study DNA has suggested that p300 auto-acetylation serves as a switch CBP/p300-dependent to regulate its arrival and departure during pre-initiation transcription complex assembly [38]. In addition, there is evidence that p300 also functions in elongation [39]. Therefore, the ac- Modulating GSTFs tivity of CBP and/or p300 and their capacity to bind with that utilise certain transcriptional regulatory proteins are subjected to CBP/p300 CBP/p300 regulation by diverse mechanisms, hence contributing to Strategies Compounds targeting specific targeting CBP/p300 CBP/p300 co-factor transcriptional specificity and plasticity. acetyl-transferase synthesis activity inhibitors Implication of CBP/p300 in respiratory epithelium Potential therapeutic approaches tumorigenesis Figure 3 CBP/p300 may contribute to respiratory epithelium car- Most of the described tumour-related mutations in CBP/ cinogenesis via multiple routes. Potential strategies for therapeutic p300 result in truncation of the p300 protein. In majority of interventions are indicated (currently tested regimens mostly target the cases, the second allele was inactivated through deletion CBP/p300 acetyl-transferase activity; thicker arrow). BTM, basal (loss of heterozygosity (LOH)), silencing (hemizygosity) transcriptional machinery; GSTF, gene-specific transcription fac- or a different (compound heterozygosity). These tor. findings have qualified p300 as a classical tumour-sup- pressor gene, but with a low detected mutation rate in cell lines [40]. It is currently less clear whether CBP should also be classified as a tumour-suppressor gene. However, the high prevalence of malignant tumours among RTS pa- tients along with the fact that both CBP and p300 proteins coexistence of CBP and p53 mutations, which suggests that are targets of transforming viruses suggest that disruption CBP gene alterations might contribute to lung carcinogen- of CBP function contributes to carcinogenesis [11]. In esis by distorting pathways other than those engaging p53. lung cancer, LOH has been frequently detected in diverse Regarding p300, somatic mutations have been identified in chromosomal regions, albeit relatively few targeted tumour several types of [43], but their prevalence in lung suppressor genes (including p53, Rb, p16 and FHIT) have cancer is unknown. The notion that CBP and p300 might been identified [41]. Recently, it was shown that the CBP play different roles in diseases such as lung tumorigenesis is gene is genetically altered in almost 15% of lung cancer supported by the observation that reintroduction of p300 but cell lines and 5% of primary lung tumours [42]. Thus, not CBP was able to suppress the growth of p300-deficient point mutations and homozygous deletions of the CBP carcinoma cells [44]. gene might be involved in the pathogenesis of a subset of Since mutations in CBP/p300 are relatively uncommon, lung carcinomas (Figure 3). Interestingly enough, these CBP/p300 might contribute to lung carcinogenesis through CBP mutations are not clustered in the catalytic (acetyl- alternative mechanisms (Figure 3). The cell-cycle apparatus transferase) region but are dispersed throughout the entire acts as a dominant controller “supervising” the cell fate, gene, indicating that the biological effects of such mutations and its deregulation represents an imperative step during are diverse [42]. Another important aspect is the observed malignant transformation. CBP/p300 are shown to associate www.cell-research.com | Cell Research npg CBP/p300 and lung cancer 328 with the cyclin E/Cdk2 complex [45]. Although the precise has been shown to be strongly associated with lung carcino- role of CBP/p300 in regulating cell proliferation remains genesis [55]. The ability of E2F-1 to stimulate transcription elusive, existing evidence suggests that they are negative appears to be subjected to multiple regulations including modulators of the cell cycle. The fact that these proteins co-activation by CBP/p300 and reversal of Rb-mediated are targets of viral oncoproteins also suggests their impor- repression through Rb phosphorylation [55]. At a molecular tance in cell-cycle regulation, such as the control of DNA level, the Cdk-stimulated interaction of CBP/p300 with synthesis and S-phase progression [46]. For example, it has E2F-1 may be involved in irreversibly committing cells to been shown that the p300/CBP-p/CAF protein complex can cell-cycle progression [56]. Interestingly, although E2F-1 arrest cell cycle and might regulate target genes that are has been shown to be acetylated in vitro by both p/CAF

involved in the control of the G1/S transition, such as p21 and CBP/p300 at the same lysine residues, a specific role [46]. In vitro models have also suggested that sequestration for p/CAF in acetylation-induced stabilisation of E2F-1 in of CBP/p300 by viral oncoproteins has the general effect response to DNA damage was recently reported [57]. of modulating transcription through affecting transcrip- One of the major tasks of CBP/p300 is the cross-coupling tion factors that normally utilise these co-activators [46], of distinct gene-expression programs in response to various while the recently recognised link between CBP/p300 and stimuli [28]. However, CBP/p300 levels in vivo are consid- the anaphase-promoting complex/cyclosomeE3 ubiquitin ered stoichiometric and apparently cannot simultaneously ligase has provided new insights into the roles of these support its various functional activities. Thus, CBP/p300 proteins in the control of both cell cycle and transcription over-expression or their preferential usage by certain [45]. “hyperactive” transcription factors, or a combination of CBP/p300 are cardinal transcriptional co-regulators both, could contribute to unopposed cellular proliferation responsible for the proper function of a gamut of signal- as well as apoptosis inhibition during lung carcinogenesis. ling cascades. To this end, the growth-suppressing effect Recently, CBP over-expression at the very early stages of of CBP/p300 might be well explained by their ability to respiratory epithelium carcinogenesis has been documented augment p53-mediated transcription [47]. The p53 tumour [28]. This observation has led to a “step-wise” model in suppressor gene is the most commonly mutated gene in which CBP over-expression accompanied by upregulation human cancers and is frequently found to be dysregulated of members of the activator protein-1 (AP-1) family and in lung pre-malignant and malignant lesions [48]. A major a gradual downregulation of the retinoid acid receptor β function of p53 is to activate genes engaged in the response might favour lung tumour progression and proliferation to DNA damage, such as murine double minute 2 (mdm2), [58]. p21, cyclin D1 and Bax [47]. Following DNA damage, This model is in accordance with clinical observations p53 is activated by kinase-mediated phosphorylation as that have identified cyclin D1 over-expression, which is well as by acetylation at specific residues by CBP/p300 AP-1 dependent, as a frequent event in human lung tumours [49], resulting in increased stability of the p53-CBP/p300- [59]. Intriguingly, recent studies have demonstrated that DNA complex. Furthermore, CBP/p300 is required for cyclin D1 could control transcription factor activity by p53-mediated transactivation of target genes through their directly interacting with and repressing the transactivation co-activator function and through local histone acetylation capacity of p300 [60]. Nevertheless, the mechanisms by [50]. It has also been suggested that the association of p53 which cyclin D1 regulates a variety of cellular functions are with CBP/p300 might account for p53-mediated negative not fully understood. Another gene that merits discussion in regulation of genes whose promoters lack a suitable p53 consideration of lung carcinogenesis is cyclooxygenase-2 binding site [51]. Interestingly, CBP/p300 also contribute (COX-2). It has been shown that growth factor-induced to controlling p53 stability by regulating its ubiquitina- COX-2 transcription is mediated through the Ras-MAPK tion and degradation, through both Mdm2-dependent and signalling pathway and through subsequent activation of Mdm2-independent mechanisms. Degradation of p53 is AP-1 [61]. COX-2 is an inducible during carci- known to be mediated by a ternary complex comprising nogenesis, and many experimental and clinicopathological p53, Mdm2 and CBP/p300 [52]. Recently, the CH1 domain studies have revealed that COX-2 over-expression is as- of CBP/p300 was found to display activity sociated with respiratory epithelium tumorigenesis through towards p53, and therefore, CBP/p300 could also play a proliferation enhancement, apoptosis inhibition and trig- direct role in p53 degradation [53]. gering of angiogenesis [62]. In this respect, it is important The E2F family of transcription factors play a pivotal to note that CBP/p300 are the predominant co-activators role in regulating cell cycle progression and apoptosis [54]. in COX-2 transcriptional activation [63]. In mammals, the E2F family has six different members. The Collectively, the accumulating evidence indicates that best-characterised member is E2F-1 and its over-expression CBP/p300 function as general co-regulators of a variety

Cell Research | www.cell-research.com Michalis V Karamouzis et al. npg 329 of transcription factors that could have either tumour-sup- parental compound Lys-CoA have recently been identified pressing or tumour-enhancing properties. The abundance and are currently being evaluated [72]. of CBP/p300 and the specific interaction mode between High-throughput screening of random chemical librar- CBP/p300 and these various factors as dictated by the ies for specific inhibitors of CBP/p300 acetyl-transferase specific cell type and cellular context are likely of critical activity is another way of identifying compounds that could importance in determining how CBP/p300 might modulate then be further modified by medicinal chemistry method- cell physiology/pathology and regulate disease pathogen- ologies to develop drugs suitable for clinical application. esis such as lung carcinogenesis. Recently, the first naturally occurring acetyl-transferase inhibitor, anacardic acid, was found. This substance inhibits The potential of modulating CBP/p300 in lung cancer very effectively, in a non-competitive manner, the activity therapeutics of both p300 and p/CAF [73], and it has been also shown to increase in vitro the sensitivity of tumours to radiation In theory, CBP/p300-mediated signal propagation dur- therapy [74]. By using this molecule as a synthon, a syn- ing respiratory epithelium tumorigenesis could be modu- thetic amine derivative of anacardic acid (CTPB) has been lated by diverse strategies (Figure 3). For instance, in the generated [75]. However, again cells were impermeable case when their over-expression constitutes the problem, or poorly permeable to both anacardic acid and CTPB. antisense oligodeoxynucleotides and RNA interference Nevertheless, these and other natural products might offer approaches could be used to reduce their production [64, valuable “probes” for identifying potential clinically effec- 65]. Targeting protein-protein interfaces has emerged as a tive remedies. For example, curcumin was recently shown promising anticancer approach, although many theoretical to exert a specific inhibitory activity towards CBP/p300 caveats have to be tackled [66]. Moreover, new technolo- [76]. The first cell permeable acetyl-transferase inhibitor gies have recently emerged to selectively block the function has also been reported. It is garcinol, a polyisoprenylated of critical transcription factors (e.g. structure-based rational benzophenone derivative of Garcinia indica fruit rind, and design of decoy oligonucleotides) [67]. has demonstrated potent inhibitory activity towards histone Being one of the key involved in post-transla- acetyl-transferases (HATs) both in vitro and in vivo [77]. In tional modifications, the acetyl-transferases CBP/p300 hold addition, a series of isothiazolone-based acetyl-transferase crucial roles in the causal relationship between dysfunction interfering agents were recently reported as being potential of the acetylation/deacetylation equilibrium and respira- small-molecular-mass inhibitors for acetyl-transferases tory epithelium carcinogenesis. During the last decade, [78]. a number of HDAC inhibitors have been identified that induce apoptosis in cultured tumour cells and have entered Conclusion clinical testing [68]. Pharmacologic inhibition of HDACs might restore the distorted epigenetic network and have Developing an integrated picture of the role of CBP/p300 therapeutic effect throughout the carcinogenesis process. in lung carcinogenesis is a challenging task that awaits fur- A proof of principle of this assumption was the recent ap- ther exploration. CBP/p300 are considered multi-functional proval of the HDAC inhibitor suberoylanilide hydroxamic transcriptional co-activators participating in a broad spec- acid for patients with progressive, persistent or recurrent trum of intracellular processes under normal and pathologic forms of cutaneous T-cell lymphoma [68]. conditions. However, many questions remain unanswered. Although substantial progress has been made in the Further genetic and functional studies of CBP/p300 would study of HDAC inhibitors, very little has been achieved aid at unravelling their prominent activities, thus generat- in the area of acetyl-transferase inhibitors. Long before, ing new options for intervention during the formation of polyamine-CoA conjugates were found to inhibit the lung tumours. acetyl-transferase activity of cell extracts [69]. Availabil- Acetylation is a feature of active genes and its inhibi- ity of recombinant acetyl-transferases (p300 and p/CAF) tion in vivo would repress majority of the genes, including rendered it possible to synthesise and test more targeted those that are aberrantly expressed. Therefore, a systematic and specific inhibitors, Lys-CoA for p300 and H3-CoA for investigation of the effect of compounds targeting acetyl- p/CAF [70]. The major problem with these compounds was transferase activity on normal and cancerous cell lines is a their lack of cellular permeability. In an effort to overcome prerequisite to define their potential utility in lung cancer this limitation, truncated derivatives were designed, syn- therapeutics. Further modifications of these compounds thesised and assessed as p300 inhibitors, with, however, in conjunction with continued research for new molecules disappointing results [71]. 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